3 | DISCUSSION
For ultrasound-induced endosomal escape, both destabilization of the
endosomal membrane and protein release from the carriers are required.
In this study, to meet the former requirement, PCNDs were employed as an
ultrasonically endosome-disruptive carrier based on a pioneering
study[28]. To meet the later requirement, a
disulfide-linked material for the bio-reductive release of proteins was
developed in this study. This material was confirmed to conjugate with a
model protein with the lipid coating of droplets and to release it in
response to reductive conditions in vitro (Figure 2A). Even in
living cells, the bio-reductive release of β-Gal and Sap was strongly
suggested by the hydrolysis of the fluorogenic substrate in the cytosol
(Figure 3) and enhancement in cytotoxicity (Figure 4), respectively.
Furthermore, cytosolic delivery of these two model proteins was observed
in an ultrasound-responsive manner (Figure 3 and 4). This result
indicates that endosomal escape of cargo proteins was achieved by
vaporization of PCNDs.
In our previous works, intracellular vaporization of antibody-conjugated
PCNDs was utilized for ultrasound-dependent induction of cell death to
targeted cells[30,31]. Different from the present
study, the PCNDs which included the equal mixture of PFH (boiling point
= 57 °C) and perfluoropentane (PFP, boiling point = 29 °C) was employed.
Here, employment of inner perfluorocarbons with a low boiling point
increases the size and the lifetime of bubbles after vaporization,
leading to remaining and coalescence of
bubbles[28]. Such properties of PCNDs with a lower
boiling point are desirable in intracellular vaporization for enhancing
cytotoxicity, and therefore, the PCNDs including the PFH-PFP mixture was
employed in the previous works[30,31]. In the
present study for cytosolic protein delivery, the PCNDs including PFH
only was employed for suppressing cytotoxicity of their vaporization.
Actually, in β-Gal delivery, no significant cell damage was observed in
the microscopic images one hour after ultrasound exposure (Fig. 3), as
observed in the pioneering study in which the PCNDs including PFH only
were used[28]. This result is quite different from
the images in our previous works in which cells were disrupted and
detached from the dish surface by PCND
vaporization[30]. However, in Sap delivery, the
vaporization of PCNDs without carrying Sap caused non-negligible
cytotoxicity after incubation for 48 h (Figure 4). In our previous work,
the cell viability after vaporization of PCNDs was influenced by the
PCND concentration during cellular uptake and the experimental setup for
ultrasound exposure[31]. Further optimization of
these conditions is required to manipulate cell functions without
causing cell damage. Thus, although there is a room for improvement, we
have demonstrated a proof-of-principle that ultrasound-dependent
cytosolic protein delivery is possible using chemically functionalized
PCNDs.
In conclusion, the protein-conjugated PCNDs could be used for
ultrasound-induced cytosolic protein delivery. Through the development
of a thiol-reactive lipid coating, cargo proteins were carried on the
droplets through a bio-reductive disulfide linkage. When the cargo
protein had no thiol group, it was conjugated with the droplets through
reversible thiolation of the amine groups. In principle, the proposed
method can be applied to any protein. These protein-conjugated PCNDs
were taken up into living cells, and after ultrasound exposure, the
cargo proteins functioned in the cytosol in an ultrasound-induced
manner, probably because of endosomal escape by the vaporization of the
droplets in the endosomal pathway. In this study, by utilizing an
anti-cancer antibody as the molecular device for intracellular delivery,
protein-conjugated PCNDs were accumulated in cancer cells. Similarly,
according to the objectives, proteins can be selectively delivered into
the cytosol of various targeted cells by employing the desired targeting
ligands. Furthermore, based on the double targeting effect of both
ultrasound and the ligands, the current method of cytosolic delivery is
expected to meet a wide variety of demands in fundamental studies and
therapeutic uses.